This invention relates to a method for the detection of the presence and concentration of compounds in a liquid medium and, in particular, to an improvement in immunoassay techniques for determining the presence and concentration of protein antigens, such as drugs, hormones and the like in a liquid sample.
The art has long recognized the need for analytical methods and apparatus for determining the presence of a particular compound or class of compounds in a liquid medium, and a variety of techniques has been developed to accomplish this aim. Particularly important areas for such methodology lie in the field of medicine wherein, for example, information regarding the presence of a particular compound in a patient is utilized as a diagnostic tool for determining various physical conditions or abnormalities. Methods for analyzing blood or urine samples for the presence of glucose, cholesterol or urea are well-known examples of these techniques.
Much of the general utility of methods for determining merely the presence of particular compounds in a liquid system, e.g., a biological fluid, is limited where accurate quantitative analysis of minute amounts or concentrations of the compound in the fluid is required. Accordingly, techniques have also been developed for achieving this aim. Two such techniques are those known as radioimmunoassay (RIA) and enzyme-linked immunoassay (ELIA), having particular applicability in the determination of the concentration of drugs or hormones in a biological fluid either in connection with treatment of a patient or in the field of forensic medicine.
The RIA and ELIA techniques utilize antibodies developed specifically for a particular compound (antigen) whose presence is to be determined. The antibody reacts with (i.e., becomes bound to) only its specific antigen and, hence, displays the specificity required for accurate quantitative determinations. In practice, the antibody prepared against, for example, amphetamine, is added to a serum sample to be analyzed. Additionally, a known quantity of amphetamine which has been labelled (i.e., coupled to a signal emitter such as radioactive tritium for the RIA technique or an enzyme-linkage whose presence can be determined spectrophotometrically for the ELIA technique) is also added to the sample. Competitive and/or non-competitive displacement then takes place over a period of time wherein both the labelled antigen and the antigen to be determined, present in the sample fluid, compete for attachment to the antibody. A material is then added which will quantitatively remove all antigen from the sample which has not been bound to antibody. The remaining fluid, containing the antibody and any bound antigen, is then transferred to an appropriate device (e.g., a scintillation counter or spectrophotometer) for determining the presence of labelled antigen therein. The quantity of labelled antigen found is a measure of the amount of unlabelled antigen in the biological sample tested. Thus, for example, if the sample contained no antigen, all the labelled antigen originally added to the sample would be removed with the antibody as would be indicated by a radiation count or optical density equal to that obtained from the known quantity of labelled antigen added to the sample at the beginning of the test. Alternatively, a determination that less than the original amount of labelled antigen now appears with the antibody indicates that antigen was present in the sample in an amount correlatable to the decreased amount of labelled antigen found with the antibody. In an alternative procedure, after the above-noted competitive attachment between labelled and unlabelled antigen has taken place, the antibody is removed from the liquid system by protein precipitation and it, rather than remaining fluid, analyzed by the appropriate method to determine the amount of labelled antigen thereon.
Variations of the RIA and ELIA techniques exist where the antibody is added to the sample already in the form of a complex with its labelled antigen. The same methods as above-described are then utilized to determine the quantity of labelled antigen, if any, displaced from the antibody as an indication of the presence of unlabelled antigen in the sample.
A recent advance in the field of RIA is described in papers authored by L. Wide, et al., appearing at Journal of Immunochemistry, 4, 381 (1967); Biochim. Biophys. Acta. 130, 257 (1966); and Acta Endochrinol., 63, Suppl. 142, 207 (1970). According to this method, radioimmunosorbentassay (RISA), the antibody and its labelled antigen are complexed or affixed to a solid support material, e.g., CNBr-activated Sephadex. This complex is then added to the fluid sample to be tested in the manner as above-described. This method provides an improved means for separating the antibody from the sample after the competitive displacement has occurred, e.g., the solid support material containing antibody, labelled antigen and antigen from the sample, if any, can simply be removed in toto from the test vial. This material, or the fluid remaining after removal of the supported material from the sample, can then be tested for its radio-activity to determine the quantity of antigen present in the original sample.
A parallel advance in the ELIA technique was first described in the work of Catt and Tregear, "SCIENCE," 158, 1570 (1967) and further elucidated by van Weeman and Schuurs, "Immunochemistry," 21,667 (1975) and Engvall and Jonsson, "Biochem. Et Biophysica Acta," 251, 427 (1971). In this method, enzyme-linked immunosorbentassay (ELISA), the antibody is coupled to a solid immunosorbent, for example, Sepharose. The immunosorbent is then incubated in a solution containing the enzyme-linked antigen and the sample to be analyzed. The antigen conjugated to an appropriate enzyme is analogous to the radio-labelled antigen in the RIA technique. As with all immunoassays, a competitive binding of the antigen tagged with the enzyme and the antigen present in the sample takes place with the antibody. After the appropriate incubation period, the immunosorbent is removed. The fluid, containing the remaining free enzyme-linked antigen is then reacted with a suitable color developer, for example, o-phenylaniline diamine and hydrogen peroxide solution to produce a colorimetric reaction for reading optical densitites in a spectrophotometer.
Although the RIA and ELIA techniques and the recent advancements therein provide a highly accurate, quantitative determination for specific antigens, a major disadvantage resides in the inability to conveniently test for the presence of more than one antigen at any one time. Thus, it is not presently possible to determine, for example, the quantitative presence of the common classes of drugs--amphetamines, barbiturates and opiates--in one test since there is no means for distinquishing the particular antibodies to which these drugs would report. One recent attempt in this regard is reported by Cleeland, et al., Clinical Chemistry, 22/6, 712-725 (1976) wherein a triple assay test for the three above-mentioned drugs is described. In this method, the three labelled antigens and their antibodies are mixed and contacted with a single biological fluid sample. However, the test is only effective in determining the complete absence of any of the drugs. Any positive indication of drug presence cannot distinguish which particular drug or drugs are contained in the sample. Hence, where a positive indication is found, the three assays must then be performed individually to determine exactly which drug or drugs caused the positive response.
A very definite need exists, therefore, for a method for immunoassay determinations capable of performing multiple assays to detect, in one test, the quantitative presence of more than one antigen.